IEF and 2D Protein Standards

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Overview

IEF Standards

IEF standards allow dependable and reproducible pI calibration in IEF gels. IEF standards consist of a mixture of nine native proteins with isoelectric points ranging from 4.45 to 9.6. Five of the nine proteins are naturally colored to provide continuous monitoring of the focusing process. IEF standards are for use in analytical acrylamide and agarose isoelectric focusing gels under native conditions. The mixture is provided in a stable aqueous solution, and no reconstitution or dilution is required prior to use.


Constituent Proteins of IEF Standards*
Protein Color pI
1. Cytochrome c
Red
9.6
2. Lentil lectin (3 bands)
7.8, 8.0, 8.2
3. Human hemoglobin C
Red
7.5
4. Human hemoglobin A
Red
7.1
5. Equine myoglobin (2 bands)
Brown
6.8, 7.0
6. Human carbonic anhydrase
6.5
7. Bovine carbonic anhydrase
6.0
8. β-lactoglobulin B
5.1
9. Phycocyanin (3 bands)
Blue
4.45, 4.65, 4.75

* Because the IEF standards are in native form, they cannot be used with reducing or denaturing agents such as urea, β-mercaptoethanol, or dithiothreitol. For calibration or IEF tube gels containing urea, use 2-D SDS-PAGE standards.

IEF standards strained with IEF gel stain. Run on Criterion™ (left) and Ready Gel™ IEF gels (right).

IEF standards for accurate pI calibration of native IEF gels.
A. 5 µl of the standards were stained with Coomassie Blue R-250 dye and crocein scarlet
B. 5 µl of the standards were left unstained

2-D SDS-PAGE Standards

These unique protein standards calibrate the pI and molecular weight of the proteins in 2-D SDS-PAGE applications. The standards consist of seven reduced, denatured proteins that can be run internally on the same gel as the sample, or alone to test the ampholyte mixture and the reproducibility of the gels. Protein spots can be visualized with silver or Coomassie Blue staining. No dilution of the standards is required.

Two-dimensional electrophoretic protein pattern of 2-D SDS-PAGE standards

Two-dimensional electrophoretic protein pattern of 2-D SDS-PAGE standards. The standards (2.5 μl) were run on 7 cm ReadyStrip™ IPG strips, then in the Mini-PROTEAN® II cell. For method details, see Klose (1975), Klose and Feller (1981), and Jungblut and Seifert (1990). The gel was stained with Bio-Rad’s silver stain kit.

Using Precision Plus Protein™ Standard Plugs

Step 1
Break and twist off one end of the plastic plug casing.

Step 2
Break off an individual plastic plug from the casing by placing pressure on the flat side to break the junction.

Step 3
Complete the break by twisting off the individual plastic plug casing.

Step 4
Snap off the attached spatula.

Step 5
Use the spatula to push the plug out of the plastic casing.

Step 6
Use the spatula to slide the plug outward and on to the top plate of your gel.

Step 7
Slide the plug all the way down the bottom of the well next to IPG strip. Then add an agarose overlay.



Second dimension SDS-PAGE gel run with a mouse brain sample, Precision Plus Standards Plugs, and stained with Bio-Safe™ coomassie G-250 stain.

Precision Plus Protein™ Standard Plug Run on a PROTEAN Plus Gel

Precision Plus Protein Standard plug on a 12% PROTEAN Plus precast gel. Sample is mouse liver.

IEF Standards

Isoelectric focusing (IEF) protein standards allow dependable and reproducible pI calibration in native polyacrylamide or agarose IEF gels. Our IEF protein standards are a mixture of nine native proteins with pI ranging from 4.45 to 9.6. Five of the nine proteins are naturally colored to aid in monitoring the focusing. These standards are provided in a stable aqueous solution, and need no reconstitution or dilution prior to use.

Constituent Proteins of IEF Standards*

Protein Color pl
1. Cytochrome c Red 9.6
2. Lentil lectin (3 bands) 7.8, 8.0, 8.2
3. Human hemoglobin C Red 7.5
4. Human hemoglobin A Red 7.1
5. Equine myoglobin (2 bands) Brown 6.8, 7.0
6. Human carbonic anhydrase 6.5
7. Bovine carbonic anhydrase 6.0
8. β-Lactoglobulin B 5.1
9. Phycocyanin (3 bands) Blue 4.45, 4.65, 4.75

* Because the IEF protein standards are in native form, they cannot be used with reducing or denaturing agents such as urea, β-mercaptoethanol, or dithiothreitol. For calibration of IEF tube gels containing urea, use 2-D SDS-PAGE standards.

2-D SDS-PAGE Standards

These unique protein standards provide calibrated references for the pI and molecular weight of proteins in 2-D SDS-PAGE applications. They consist of seven reduced, denatured proteins that can be visualized with silver or Coomassie Blue staining. No dilution is required.

Constituent Proteins of 2–D SDS-PAGE Standards

Protein pl MW
1. Hen egg white conalbumin  6.0, 6.3, 6.6 76,000
2. BSA  5.4, 5.5, 5.6 (empirically determined) 66,000
3. Bovine muscle actin 5.0, 5.1 (empirically determined) 43,000
4. Rabbit muscle GAPDH
8.3, 8.5 36,000
5. Bovine carbonic anhydrase  5.9, 6.0 31,000
6. Soybean trypsin inhibitor  4.5 21,500
7. Equine myoglobin
7.0 17,500

Precision Plus Protein™ Standard Plugs

Precision Plus Protein Standards are available cast into 1 mm thick agarose plugs for easy storage, handling, and loading. They come in easy-to-use snap-off molds in packs of 24 (one application per plug), allowing quick and clean loading of molecular weight standards on any gel. These plugs can be used on vertical 2-D gels with no reference well.

These plug protein standards are unstained. Loading concentrations have been optimized for staining with SYPRO Ruby, Silver Stain Plus™, or Bio-Safe™ Coomassie Blue.

Features and Benefits of Precision Plus Protein Standard Plugs

  • Precast for ease of use — just snap, twist, and load the plug onto a gel
  • Absolute molecular weight accuracy, confirmed by mass spectrometry
  • Ideal ten-band range from 10 kD to 250 kD, with 3 high-intensity reference bands (25 kD, 50 kD, and 75 kD) for instant gel orientation
  • Molecular weights that never change, so band sizes are easy to remember
  • High r2 value, >0.995, allowing accurate molecular weight determination
  • Known concentration of protein in each band, allowing quantitation capability
  • Strep-tag amino acid sequence for detection and molecular weight determination on western blots
MW size range Varies by standard
Stain type Unstained
Number of applications Depends on staining method
Volume IEF: 250 µl; 2D: 500 µl
Shelf life 12 months at –20°C
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IEF Standards

1610310
250 μl, pI range: 4.45–9.6, phycocyanin, β-lactoglobulin B, bovine and human CA, equine myoglobin, human hemoglobin A and C, lentil lectin, and cyt C

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$120.00
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2-D SDS-PAGE Standards

1610320
500 μl, 17,500–76,000 MW range, pI 4.5–8.5, unstained mixture of myoglobin, trypsin inhibitor, CA, GAPDH, actin, BSA, and conalbumin

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$195.00
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Precision Plus Protein™ Standard Plugs
Precision Plus Protein™ Standard Plugs, unstained, Strep-tagged, recombinant

1610378
Pkg of 24, 1 mm thick agarose plugs containing ten Strep-tagged, recombinant proteins (10–250 kD), including three reference bands

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$120.00
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Number Description Options
2414 The Little Book of Standards, Rev D Click to download [ Add to Cart (Free) ]
3118 Protein Standards for Electrophoresis and Blotting Brochure, Rev D Click to download [ Add to Cart (Free) ]
2998 Protein Standards Application Guide, Rev G Click to download [ Add to Cart (Free) ]
LIT396 Instruction Manual, IEF Standards, Rev F Click to download
4110064 Instruction Manual, 2-D SDS-PAGE Standards, Rev A Click to download
4117684 Instruction Manual, Precision Plus Protein™ Standard Plugs, Unstained, Rev C Click to download